Apoptotic cell death that is regulated by Bcl-2 family proteins plays a significant role in acute and chronic neuropathologies such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. A specific role for the Bcl-2 family protein BAD in inducing apoptosis has been elucidated for hippocampal neurons treated with glutamate in vitro and for neurons of the spinal cord following traumatic injury in vivo. BAD is a target for multiple levels of post-translational modification, including phosphorylation, proteolytic cleavage, and intracellular relocalization. The manner in which these modifications regulate the function of BAD within neurons, however, remains incompletely defined. This laboratory has made the novel observation that BAD can be either anti-apoptotic or pro-apoptotic depending on the cellular context. Defining the cellular mechanisms of regulation that determine BAD function therefore holds considerable promise for both neuroprotective and anti-cancer interventions.Specific Aim 1 will use BAD knock-in mutant mice and cell culture models to test the hypothesis that phosphorylation, cleavage, and mitochondrial targeting of BAD regulate the conversion of BAD from an anti-apoptotic to a pro-apoptotic signaling molecule. Specific Aim 2 will use cell culture models and immunofluorescent imaging techniques to identify the step in the cell death pathway that is inhibited by BAD. Specific Aim 3 will use primary cortical cultures and mice to identify differences in BAD post-translational modification and binding interactions that differ during maturation when BAD undergoes reversal of function. Affinity-purification and yeast-two-hybrid screening will be used to search for novel BAD regulatory proteins that are differentially expressed during development.